Imaging-Based High-Throughput Screening Assay To Identify New Molecules with Transmission-Blocking Potential against Plasmodium falciparum Female Gamete Formation

In response to a call for the global eradication of malaria, drug discoveryhas recently been extended to identify compounds that prevent the onward transmission of the parasite, which is mediated by Plasmodium falciparum stage V gametocytes. Lately, metabolic activity has been used in vitro as a surrogate for gametocyteviability; however, as gametocytesremain relatively quiescent at this stage, their ability to undergo onward development ( gameteformation) may be a better measure of their functional viability. During gameteformation, female gametocytesundergo profound morphological changes and express translationally repressed mRNA. By assessing female gametecell surface expression of one such repressed protein, Pfs25, as the readout for female gametocytefunctional viability, we developed an imaging-based high-throughput screening (HTS) assay to identify transmission-blocking compounds. This assay, designated the P. falciparum female gametocyteactivation assay (FGAA), was scaled up to a high-throughput format ( Z′ factor, 0.7 ± 0.1) and subsequently validated using a selection of 50 known antimalarials from diverse chemical families. Only a few of these agents showed submicromolar 50% inhibitory concentrations in the assay: thiostrepton, methylene blue, and some endoperoxides. To determine the best conditions for HTS, a robustness testwas performed with a selection of the GlaxoSmithKline Tres CantosAntimalarial Set (TCAMS) and the final screening conditions for this library were determined to be a 2 μM concentration and 48 h of incubation with gametocytes. The P. falciparum FGAA has been proven to be a robust HTS assay faithful to Plasmodium transmission-stage cell biology, and it is an innovative useful tool for antimalarial drug discoverywhich aims to identify new molecules with transmission-blocking potential.